Twenty microliters of blood in 96-well plates were incubated for 2 h with 6 x 104 CFU L. monocytogenes in 130 μl RPMI without additives. Plates were shacked for 1 min at 400 rpm using a microplate shaker (Edmund Buhler, Bodelshausen, Germany) and serial dilutions of blood were plated on Columbia III Agar with 5% Sheep blood medium (BD Biosciences). Plates were incubated for 24 h at 37°C, and colonies were enumerated. Given that L. monocytogenes is an intracellular Gram-positive bacterium, we verified that similar CFUs were obtained using native blood and lysed blood samples (native/lysed x 100: 106.9 ± 15.1%, n = 8, P > 0.1). One hundred thousand PMNs or monocytes in 1.5 ml tubes were incubated for 2 h with 103 CFU L. monocytogenes in 200 μl RPMI containing 2% murine plasma. Tubes were centrifuged 10 min at 4,100 × g. Pellets were resuspended in 200 μl sterile water and incubated for 10 min to lyse cells. Spleens were homogenized with an UltraTurrax homogenizer (IKA-Werke, Staufen, Germany). CFUs were determined as described above.
Columbia 3 agar
Manufactured by BD
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Columbia III agar is a general-purpose microbiological culture medium used for the growth and isolation of a wide range of bacterial species. It provides a nutritious environment that supports the cultivation of both fastidious and non-fastidious organisms.
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4 protocols using columbia 3 agar
1
Quantitative Listeria Infection Assay
2
Preparation of Bacterial and Yeast Suspensions
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Most bacterial and yeast strains used in this study (Tables A.1–A.3 ) were selected according to the most prevalent strains isolated in clinical urine samples in 2014 at the University Hospital of Lausanne (CHUV), Switzerland. The strains were grown on Columbia agar with 5% sheep blood (Columbia III agar; BD, Franklin Lakes, NJ, USA) at 37 °C in normal atmosphere or in 5% CO2 atmosphere incubators. Colonies of each bacterial species were utilized to prepare a bacterial suspension in saline solution adjusted to a 0.5 McFarland turbidity measured with a DensiCheck densitometer instrument (bioMérieux, Marcy l'Etoile, France). The exact bacterial concentration corresponding to a 0.5 McFarland were assessed for each bacterial and yeast species and for each experimental run by measuring the colony forming units (CFU) on Columbia agar with 5% sheep blood (Table A.1 ). Different concentrations of monomicrobial suspensions were prepared by doing serial 10-fold dilutions in saline solutions, ranging from 1 to 10−5.
3
AmpC and ESBL Detection in Bacterial Isolates
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Deep-frozen samples of the selected strains were recultured on Columbia III agar (BD Diagnostic Systems, Sparks, MD, USA) or blood agar (Media production, Elizabeth-Tweesteden Hospital, Tilburg, the Netherlands) prior to testing. Strains were tested using Etest (bioMérieux, Marcy-l’Étoile, France) to determine the MICs of cefotaxime, ceftazidime and cefoxitin. Etests were placed on Mueller–Hinton (Oxoid Ltd, Altrincham, Cheshire, England) culture plates, which were placed in the oven within 15 min and incubated for 16–20 h under an O2 atmosphere at 36°C. Exact MIC values were noted. The presence of AmpC was phenotypically confirmed using the AmpC Confirm Kit (Rosco Diagnostica A/S, Taastrup, Denmark) according to the manufacturer’s guidelines. A second phenotypical confirmation with the D68C AMPC + ESBL detection set (MAST Group Ltd, Bootle, UK) was performed according to the manufacturer’s guidelines. From both confirmation tests the zone inhibition differences, measured in millimetres, were recorded for further use.
4
Staphylococcus aureus Muscle Infection Model
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S. aureus (substrain 25923, ATCC, Manassas, VA, USA) were seeded in Columbia III agar containing 5% sheep blood (BD Biosciences, San Jose, CA, USA) and incubated overnight at 37°C. Next, in duplicate, 3 to 5 colonies S. aureus were transferred to 4.5 ml of brain heart infusion (BHI) broth (BD) in 15-ml falcon tubes and incubated overnight at 37°C. The following day, the suspension was spun down at 1,000g, and the pellet was resuspended in 5 ml of NaCl 0.9% (Laboratoire Aguettant, Lyon, France); subsequently, the content of both tubes were pooled (10 ml, 1 × 109 colony-forming units (CFU)/ml). This suspension was transported at –80°C on dry ice and thawed 1 h later right before use. The bacterial suspension was mixed 1:1 v/v with heparinized homologous blood collected by heart puncture from isoflurane-anesthetized donor mice. The exact CFU count was calculated from a titration series of the sample, which was incubated overnight on agar at 37°C. Mice were anesthetized using isoflurane, and S. aureus (50 µl, with CFUs ranging from 0.8 × 107 to 4.5 × 107) was injected in the left calf muscle 48 h before radiolabeled antibody injection for the infection and subsequent inflammatory response to develop sufficiently (25 (link)).
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